Field of the Invention
The subject matter of the application is in the field of biochemistry (immunology) and medicine and it relates to adoptive transfer therapy using tumor-specific allogeneic cells.
Background Description
Adoptive cell therapy (ACT) is a procedure in which therapeutic lymphocytes are administered to patients in order to treat either viral infection or cancer [1, 2]. This approach entails the ex vivo generation of tumor specific T cell lymphocytes and infusing them to patients. In addition to the lymphocyte infusion the host may be manipulated in other ways which support the take of the T cells and their immune response, for example, preconditioning the host (with radiation or chemotherapy) and administration of lymphocyte growth factors (such as IL-2) [1, 3, 4]. There are many methods for generating such tumor specific lymphocytes with the two main approaches being expansion of antigen specific T cells or redirection of T cells using genetic engineering [1, 5, 6]. The most notable success of ACT has been in the treatment of metastatic melanoma. In a landmark clinical trial, Dudley et al. lymphodepleted melanoma patients and then administered autologous tumor infiltrating lymphocytes expanded ex vivo, concurrently with IL-2 achieving objective responses in over 50% of the patients [7]. These results are superior to all other therapies targeting metastatic melanoma, and it is the only form of specific immunotherapy which has been proven to confer therapeutic benefit [1].
While the results observed in melanoma are very impressive, tumor infiltrating lymphocytes (TIL) can only be isolated from melanoma or renal cancer, and therefore they cannot be used a general strategy to treat cancer [1]. Gene modification has been used to redirect lymphocytes against tumors via the use of transgenic TCR chains or chimeric receptors [1, 5, 6]. The inventors' lab has pioneered the use of antibody based chimeric receptors (chimeric antigen receptor—CAR) as a means of redirecting T cells (‘the T-body approach’) against tumor antigens [8]. The T-body is a regular T cell which expresses a TCR and a chimeric receptor, and is capable of being activated by either receptor. The original chimeric receptor was composed of a scFv fragment fused to a gamma chain [9]. A ‘second generation’ tripartite chimeric receptor (TPCR) was used, and it includes an additional signaling moiety (e.g. CD28 or CD137 or their combination) and is capable of activating naïve T cells in a co-stimulation independent manner, demonstrating its superiority over the native TCR [8]. The validity of the T-body approach has been validated in numerous pre-clinical models, demonstrating activity against hematological malignancies and solid tumors (including ovarian, prostate, breast, renal, colon, neuroblastoma and others) [2, 5, 10]. There have been a few initial clinical trials employing CAR modified T cells which failed to provide significant therapeutic benefit, but these trials mainly utilized ‘first-generation’ CAR (which lack co-stimulatory motifs in the CR) and did not include prior lymphodepletion of patients [5, 10, 11]. In a recent landmark clinical trial, Pule et al. show that EBV CTL engineered with a GD2-specific chimeric receptor persist longer in vivo and provide some therapeutic benefit against neuroblastoma, demonstrating the potential of the ‘T-body’ approach [12].
Despite these successes ACT has one major drawback: each patient receives an individually fabricated treatment, using the patients' own lymphocytes, thus limiting the practicality of ACT due to substantial technical and logistic hurdles facing its application. Ideally, one would like to transform ACT into a standardized therapy in which off the shelf, ready for use ‘universal’ allogeneic therapeutic cells could be administered to patients. By allogeneic it is meant that the cells are obtained from individuals belonging to the same species but are genetically dissimilar. The problem with using allogeneic cells is double edged. In immune-competent hosts allogeneic cells are rapidly rejected, a process termed Host vs. Graft reaction (HvG) [13, 14]. In immune-incompetent hosts allogeneic cells can overcome the host's immune system, and cause serious damage and even death, a process termed Graft vs. Host disease (GvHD) [13, 14]. In order to affect adoptive therapy using allogeneic cells one would have to overcome these problems.
HvG reaction is mediated by T, B, and NK cells. T cells can either recognize allogeneic MHC molecules directly (major histocompatibility mismatch) or alternatively they can recognize non-self peptides (derived from foreign polymorphic proteins) in the context of self MHC molecules (indirect recognition stemming from minor histocompatibility mismatch) [13, 14]. B cells can recognize any foreign protein presented on the cell membrane (be they foreign MHC molecules or other polymorphic proteins) [1,5]. In addition B cells can also recognize foreign carbohydrate moieties, namely the ABO blood group antigens (as well as other blood group antigens) [1,6]. However blood group mismatch can be easily avoided, and does not usually present a problem. NK cells recognize allogeneic cells using a completely different strategy termed ‘Missing Self’ [17]. NK cells possess receptors capable of recognizing self MHC molecules such that in the presence of syngeneic cells NK cells are inhibited [17]. Importantly NK cells express these inhibitory receptors in a variegated fashion such that not all NK express all possible inhibitory receptors [17]. The result of this expression pattern is that some NK cells are capable of ‘sensing’ the absence of a single MHC molecule [17]. In this way T and NK cells complement each, and evading one cell type invites attack from the other.
The GvHD reaction only occurs when the host's HvG reaction is impaired usually in the context of allogeneic bone marrow transplantation, but also in some experimental conditions such as the parent to F1 transplantation model [13, 14]. Donor allo-reactive T cells migrate to lymphatic organs, proliferate extensively, and then egress and attack peripheral organs [14]. The potential to cause GvHD depends on two main factors: the ability to reach the lymphatic organs, and the potential for extensive proliferation [14, 18]. The ability to reach the lymph nodes is determined by expression of the lymph homing molecules CD62L and CCR7 [19]. These molecules are expressed by naïve T cells, and central memory T cells (Tcm), but not by effector memory T cells (Tem) [20-22]. Indeed studies have shown that Tcm produce much weaker GvHD than naïve or Tcm cells, and that blocking entry into lymphatic organs can prevent GvHD [23].
Due to the hurdles facing allogeneic adoptive therapy, allogeneic cells have only been employed in a handful of studies. Prior to the instant invention, the few studies which employed allogeneic ACT did so exclusively in the context of allogeneic bone marrow transplantation (allo-BMT). The preconditioning for allo-BMT ablates the host's immune system allowing engraftment of the donor bone marrow. In this setting, there is no HvG response against the original donor, and the main problem with this therapy is the development of GvHD (which can occur even if the host and donor are MHC matched) [13, 14]. The first successful application of allogeneic ACT was accomplished through the use of donor lymphocyte infusion (DLI) in the treatment of CML following allo-BMT [24-26]. The infused donor lymphocytes attack the tumor, and are capable of causing tumor regression [26]. Unfortunately because of the inherent GvH reactivity of the donor lymphocytes GvHD is a major problem with DLI [24-26]. Beginning with that initial trial, extensive work has been done to determine the optimal cell dose and conditioning regimen needed for optimal tumor response not just in CML, but also in other hematological malignancies [24, 25]. Despite these many repeated attempts. DLI has failed to show significant efficacy in other types of hematological malignancies (AML, ALL, CLL, etc) in clinical trials, so this approach does not constitute a general strategy to target tumors [24, 25]. Since then, there have been many attempts to replace the non-specific cells used in DLI with tumor specific cells. In attempt to replace DLI with tumor specific cells, Baker et al. developed a culturing protocol which yields cells with broad tumor recognition (based on NKG2D recognition), named cytokine induced cells (CIK) [27-29]. CIK cells are generated through extended culturing protocol involving extensive proliferation in the presence of IFN-γ [27-29]. These cells exhibit broad tumoricidal activity against numerous leukemias in an MHC independent manner, and importantly cause much less GvHD after allogeneic MHC mismatched BMT than fresh T cells [27-29]. The prolonged culture required in generating these cells reduced their proliferative capacity as compared with fresh splenocytes which explains at least partially the lower level of GvHD caused by these cells [28]. While these results are promising, this approach has only shown efficacy in treating hematological malignancies and little or no efficacy against solid tumors. Another drawback is that this approach and all other approaches published prior to the instant invention rely on prior allo-BMT. This dependence on allo-BMT is problematic for two reasons: first it requires complete or nearly complete MHC matching otherwise the result is overwhelming GvHD, second even if a suitable donor is found the preconditioning regimen is associated with considerable toxicity and morbidity limiting its use in some patients (such as elderly patients). In addition these treatments are only applicable when cells are obtained from the original donor which means each patient is individually treated negating the possibility of a standardized treatment.
Two papers were recently published which employed allogeneic ACT in conjunction with syngeneic BMT. In the first study, Boni et al. used haploidentical splenocytes from transgenic TCR mice to treat large established B16 melanomas after intense preconditioning in combination with autologous BMT [30]. The rationale behind this study was that myeloablation should completely prevent the HvG reaction allowing the allogeneic T cells to attack the tumor, but concurrently exposing the host to the risk of GvHD. In this case, the use of transgenic T cells which express a monoclonal TCR prevented development of acute GvHD, while infusion of open repertoire T cells did cause acute GvHD, demonstrating that a monoclonal TCR can posses little or no allo-reactivity [30]. Therapeutic benefit using allogeneic cells was only observed when the host was completely myeloablated with 9 gray, with little or no benefit at 5 gray, a fact that the authors explained by the relatively brief persistence of allogeneic cells after 5 gray irradiation (less than 10 days) [30]. Importantly, while 9 gray irradiation facilitated enhanced persistence by allogeneic cells, they nevertheless provided inferior benefit as compared with syngeneic cells which the authors attributed to the eventual rejection of the allogeneic cells by the host [30]. In addition, the use of haploidentical cells in this model, while very challenging, nevertheless, falls short of a fully mismatched model, which means that some matching between donor and host is still needed precluding using this approach as a standardized therapy.
In the second paper. Zakrezewski et al. (Marcel van den Brink's group in collaboration with Michel Sadelain) developed a completely novel approach which entails adding gene modified T-cell precursors to syngeneic BMT in a model of minimal residual B cell lymphoma [31]. Since the T-cell precursors mature in the host's thymus they undergo negative selection and lose GvH reactivity, but unfortunately maturation in the thymus also purges any GvL reactivity they possess [31]. Transduction with a anti-CD19 chimeric receptor redirects the maturing T cells against the residual lymphoma providing a significant but modest survival advantage with no long term survivors [31].
Allogeneic ACT has been proven to be successful when practiced following allogeneic bone marrow transplantation. Proper MHC matching can limit the occurrence of severe GvHD, but unfortunately also limits the applicability of allo-BMT. Without allogeneic bone marrow transplantation the host's immune system will eventually reject all of the transferred allogeneic cells, and the rate of rejection depends on the immune-competence of the host.